Hepatitis C antigen - antibody combination assay for the early detection of infection

ABSTRACT

Described herein is an ELISA based assay for the detection of HCV infection. This new assay can detect HCV infection earlier than the currently used assays for the screening of blood for HCV infection by using a combination of HCV antigens and anti-core antibodies to capture HCV.

BACKGROUND OF THE INVENTION

[0001] An estimated 170 million people worldwide have been infected byhepatitis C virus (HCV). In the next few years, the number of U.S.deaths for HCV-caused liver disease and cancer may overtake deathscaused by Acquired Immune Deficiency Syndrome (AIDS).

[0002] The transmission of HCV seems to require blood-to-blood contact.Carrying a single strand of ribonucleic acid (RNA), HCV contains justone gene, coding for a polyprotein that is subsequently spliced into atleast 10 functional proteins. Clearly, the ability to test the bloodsupply for HCV is of great importance. Furthermore, the earlier thestage of HCV one can detect, the better.

[0003] Therefore, we have developed an ELISA based assay for thedetection of HCV infection. This new assay can detect HCV infectionearlier than the currently used assays for the screening of blood forHCV infection. Current assays are based on the detection of anti-HCVantibodies in infected blood by capturing these antibodies by HCVprotein sequences represented by recombinant proteins or peptides.

[0004] For example, the Ortho HCV 3.0 ELISA uses multiple antigens todetect anti-HCV antibodies very early in seroconversion. However, thereis a significant window period in viremic individuals before theydevelop anti-HCV antibodies. This window period can be as long as 60days. During this period the individual, though highly infective, willgo undetected by current HCV screening assays.

SUMMARY OF THE INVENTION

[0005] One object of the invention is to provide a diagnostic test thatcan detect HCV infection during the window period described above bycapturing both HCV core antigen and antibodies. We describe herein amethod for detecting HCV infection earlier than the currently usedantibodies assays. This has been accomplished by providing a test thatdetects HCV core antigen in addition to the anti-HCV antibodies.

[0006] Another object of the invention is to provide a method fordetermining the presence of HCV in a sample, comprising, contacting thesample with HCV antigens and anti-HCV core antibodies attached to asolid phase, adding a polyoxyethylene ether to said sample, detectingcaptured antigens and antibodies by adding labeled anti-human IgG andlabeled anti-HCV core antibodies and detecting the signal as anindication of the presence of HCV infection.

[0007] Another object of the invention is to provide certain detergentsthat are needed to release the HCV core antigen from the virus bydisrupting the envelope protein and/or the lipid layer.

DETAILED DESCRIPTION OF THE INVENTION

[0008] It is preferrable in a combination assay for the detergents torelease the core antigen from the virus and yet not have a negativeimpact on the ability of the HCV recombinant proteins to captureanti-HCV antibodies. In a preferred embodiment of the invention,detergents from the polyoxyethylene ethers class are used for thispurpose. Commonly available detergents in this class are: BRIJ 30,BRIJ35,56,58, 92, 96, 98,700 and MYRJ 52,59,53,45. These detergents help inreleasing the HCV core antigen from the virus but the presence of thesedetergents do not negatively impact the anti-HCV assay Certaindetergents can effect the antibody detection by either affecting therecombinant antigens coated on the solid phase or inactivating theanti-HCV antibodies in the sample. Some of the detergents like N-LaurylSarcosine used in the detection of HCV core assay destroy the ability ofHCV recombinant proteins c22-3, c200 and NS5 to detect anti HCVantibodies in Ortho HCV 3.0 ELISA.

[0009] As used herein a “sample” refers to any substance which maycontain the analyte of interest. A sample can be biological fluid, suchas whole blood or whole blood components including red blood cells,white blood cells, platelets, serum and plasma, ascites, urine,cerebrospinal fluid, and other constituents of the body which maycontain the analyte of interest.

[0010] As used herein “antigen” rerers to any antigenic substanceincluding recombinant proteins and peptides or a mixture thereof.

EXAMPLE

[0011] The following examples demonstrate the advantages and utility ofthe invention by describing a list of detergents evaluated and theresults obtained by using the above described assay. One skilled in theart would recognize that the detection of HCV antibody and antigen canbe done separately or simultaneously. These examples are meant toillustrate, but not limit, the spirit and scope of the invention.

Example 1 An Antigen-Antibody Combination Assay

[0012] HCV antigens c200-3, NS-5 and a modified core antigen C22KSN47,48 or c22KSR47L along with two anti-core monoclonal antibodies (anticore murine monoclonals Pep10,12) were coated onto microwells inphosphate buffer. Antigens were modified by modifying the DNA clonescoding for the recombinant proteins c22-3 containing the HCV1 sequence.These procedures involve site directed mutagenesis using syntheticoligonucleotides (Sambrook, Fritsh and Maniatis in Molecular Cloning, Alaboratory manual, second edition, Cold Spring Harbor Press, Chapter 15,1989). After overnight incubation the buffer containing the coatingproteins was removed and the microwells washed with phosphate bufferedsaline containing a detergent Tween 20. The antigen/antibody coatedmicrowells were then treated with BSA/sucrose solution to block all ofthe protein binding sites on the microwells. After 2-24 hours theBSA/sucrose solution was removed and the microwells air dried and storedunder a descicant.

Example 2

[0013] 100 mL of sample to be tested was diluted into 100 mL of PBSsolution containing bovine serum albumin, superoxide dismutase, yeastextract and 1% Brij 58 or Brij 35 or a mixture of both. The dilutedspecimens were pipetted into HCV antigen/monoclonal antibodies coatedmicrowells (as described in Example 1). These microwells were incubatedat 37° C. for 90 minutes. The microwells were then washed 5 times withPBS containing 0.5% Tween 20. A 200 mL solution of a murine anti-humanIgG labeled with horseradish peroxidase (HRP) and an anti-HCV coremonoclonal antibody (Anti Pep4) labeled with HRP was added. After anincubation of 30 minutes the microwells were washed 5 times with PBSTween 20. A solution of orthophenylenediamine and hydrogen peroxide wasadded to each well. After incubation in the dark for 30 minutes thereaction was stopped by adding 50 microliters of 4N sulfuric acid. Theplates were read at 495 nM. An orange color in either well indicatesthat the specimen being tested is infected with HCV.

Example 3 Effects of Polyoxyethylene Ethers on HCV Antibody or AntigenAssays

[0014] Effect of the addition of various polyoxyethylene ethers on theHCV 3.0 anti-HCV assay or HCV core antigen ELISA assay is shown below inTables 1A and 1B. In the HCV core antigen ELISA assay N-Lauryl sarcosineused in the commercial HCV core antigen was replaced by differentconcentrations of detergents in column one. In the HCV 3.0 anti-HCVassay the detergents were added to the specimen diluent used in the HCV3.0 assay. TABLE 1A HCV 3.0 Ab ELISA Base Matrix HCV Antibody ReactiveDetergent Tested 0.25%  0.125% 0.06% 0.25%  0.125% 0.06% SYN. 0.125%0.06%  0.03% 0.125% 0.06%  0.03% polyoxyethylene 2 oleyl ether Brij 920.056 0.035 0.041 1.543 1.432 1.405 polyoxyethylene 8 stearate Myrj 450.019 0.024 0.025 1.208 1.286 1.313 polyoxyethylene 5 lauryl ether 0.0330.046 0.058 1.168 1.274 1.351 polyoxyethylene 50 stearate Myrj 53 0.0440.069 0.083 1.368 1.361 1.262 polyoxyethylene 8 lauryl ether 0.064 0.0700.059 1.223 1.219 1.277 polyoxyethylene 9 lauryl ether 0.057 0.055 0.0611.312 1.260 1.225 polyoxyethylene 10 lauryl ether 0.049 0.059 0.0641.192 1.069 1.161 polyoxyethylene 10 cetyl ether Brij 56 0.053 0.0930.059 0.994 1.212 1.209 polyoxyethylene 10 oleyl ether Brij 97 0.0310.056 0.072 1.316 1.341 1.534 polyoxyethylene 100 stearate Myrj 59 0.0470.050 0.045 1.751 1.615 1.418 polyoxyethylene 20 cetyl ether Brij 580.058 0.046 0.062 1.568 1.507 1.488 polyoxyethylene 40 stearate Myrj 520.048 0.063 0.067 1.502 1.308 1.194 polyoxyethylene 100 stearyl etherBrij 700 0.043 0.045 0.052 1.600 1.482 1.503 polyoxyethylene 20 oleylether Brij 98 0.053 0.044 0.050 1.632 1.542 1.493 polyoxyethylene 23lauryl ether Brij 35 0.067 0.055 0.065 1.492 1.415 1.473 polyoxyethylene7 lauryl ether 0.065 0.105 0.100 1.151 1.458 1.497 polyoxyethylene 4lauryl ether Brij 30 0.093 0.058 0.185 0.867 1.135 1.190 polyoxyethylene3 lauryl ether 0.035 0.042 0.075 0.995 0.927 1.156 Pluronic F-127 0.0250.030 0.035 1.521 1.327 1.271 Control 0.075 1.358 (3.0 SD) (3.0 SD)

[0015] TABLE 1B HCV Antigen Protoype Base Matrix HCV Antigen ReactiveDetergent Tested 0.25%  0.125% 0.06% 0.25%  0.125% 0.06% SYN. 0.125%0.06%  0.03% 0.125% 0.06%  0.03% polyoxyethylene 2 oleyl ether Brij 920.010 0.010 0.032 0.609 0.847 0.502 polyoxyethylene 8 stearate Myrj 450.028 0.021 0.022 0.880 0.509 0.433 polyoxyethylene 5 lauryl ether 0.0020.004 0.007 0.195 0.243 0.265 polyoxyethylene 50 stearate Myrj 53 0.0140.040 0.006 0.573 0.536 0.659 polyoxyethylene 8 lauryl ether 0.009 0.0100.014 0.326 0.384 0.343 polyoxyethylene 9 lauryl ether 0.008 0.008 0.0050.360 0.379 0.212 polyoxyethylene 10 lauryl ether 0.010 0.013 0.0200.495 0.556 0.344 polyoxyethylene 10 cetyl ether Brij 56 0.004 0.0010.016 0.445 0.298 0.390 polyoxyethylene 10 oleyl ether Brij 97 0.0040.006 0.021 0.371 0.419 0.479 polyoxyethylene 100 stearate Myrj 59 0.0190.021 0.023 0.429 0.320 0.369 polyoxyethylene 20 cetyl ether Brij 580.008 0.011 0.028 0.546 0.898 0.564 polyoxyethylene 40 stearate Myrj 520.009 0.005 0.006 0.579 0.412 0.661 polyoxyethylene 100 stearyl etherBrij 700 0.016 0.023 0.019 0.294 0.307 0.319 polyoxyethylene 20 oleylether Brij 98 0.008 0.011 0.032 0.557 0.718 0.539 polyoxyethylene 23lauryl ether Brij 35 0.022 0.028 0.029 0.722 0.494 0.309 polyoxyethylene7 lauryl ether 0.009 0.009 0.016 0.228 0.289 0.309 polyoxyethylene 4lauryl ether Brij 30 −0.001 0.001 0.008 0.194 0.158 0.212polyoxyethylene 3 lauryl ether 0.004 0.005 0.013 0.106 0.168 0.162Pluronic F-127 0.013 0.013 0.012 0.324 0.285 0.268 Control 0.001 1.109(nLs) (nLs)

Example 4 Detection of HCV Core Antigen and/or Anti-HCV Antibodies

[0016] Detection of HCV core antigen or anti-HCV antibodies on platescoated with anti-core monoclonal antibodies c11-3 and c11-7 and HCVantigens c200-3, NS5 and a modified core antigen. (SOD fused proteincontaining core sequences aa10-99 with aa 47 and 48 deleted(c22KS(∇47-48). The specimens used were 4 sequential bleeds from acommercially available seroconversion panel. Core antigen was detectedusing c11-4 monoclonal antibody labeled with HRP, anti-HCV antibodieswere detected by anti-human IgG labeled with HRP. By using the twoantibodies together a cumulative signal was produced, as shown in thecolumn labeled combo in Table 2. TABLE 2 ANTI ANTI SAMPLE IgG HCV CORECOMBO NEG MEAN* 0.045 0.036 0.094 BCP 6215 1 0.007 0.135 0.193 BCP 62152 0.009 0.157 0.191 BCP 6215 3 0.014 0.231 0.270 BCP 6215 4 0.494 0.1970.614

Example 5 Detection of HCV Core Antigen and/or Anti-HCV Antibodies

[0017] Detection of HCV core antigen and/or anti-HCV antibodies onplates coated with anti-core monoclonal antibodies c11-3 and c11-7 andHCV antigens c200-3, NS5 and a modified core antigen. (SOD fused proteincontaining core sequences aa10-99 with arginine at position 47 replacedwith a leucine (c22KS (R47L)). The specimens used were 4 sequentialbleeds from a commercially available seroconversion panel. Core antigenwas detected using c11-4 monoclonal antibody labeled with HRP, anti-HCVantibodies were detected by anti-human IgG labeled with HRP. In thecombo assay the two detecting antibodies, anti core monoclonal andanti-human IgG monoclonal antibody labeled with HRP were used as amixture. TABLE 3 ANTI ANTI SAMPLE IgG HCV CORE COMBO NEG MEAN 0.0450.036 0.094 BCP 6215 1 0.007 0.135 0.193 BCP 6215 2 0.009 0.157 0.191BCP 6215 3 0.014 0.231 0.270 BCP 6215 4 0.494 0.197 0.614

Example 6 Anti-IgG Comparison

[0018] A comparison of anti-HCV reactivity of seroconverter panel inmicrowells coated with two different HCV core proteins is shown in Table4. TABLE 4 SAMPLE SOD/c22KS (Δ47-48) SOD/c22KS (R47L) NEG MEAN 0.0450.076 BCP 6215 1 0.007 0.015 BCP 6215 2 0.009 0.021 BCP 6215 3 0.0140.028 BCP 6215 4 0.494 0.498

Example 7

[0019] Comparison of core antigen detection of seroconverter panel inmicrowells coated with two different core antigens is shown in Table 5.TABLE 5 Anti-HCV Core Comparison SAMPLE SOD/c22KS (Δ47-48) SOD/c22KS(R47L) NEG MEAN 0.036 0.047 BCP 6215 1 0.135 0.224 BCP 6215 2 0.1570.189 BCP 6215 3 0.231 0.304 BCP 6215 4 0.197 0.189

Example 8

[0020] Comparison of combo reactivity of seroconverter panel inmicrowells coated with two different core antigens is shown in Table 6.TABLE 6 HCV Combo Comparison SAMPLE SOD/c22KS (Δ47-48) SOD/c22KS (R47L)NEG MEAN 0.094 0.144 BCP 6215 1 0.193 0.247 BCP 6215 2 0.191 0.226 BCP6215 3 0.270 0.328 BCP 6215 4 0.614 0.658

We claim:
 1. A method for determining the presence of HCV in a sample,comprising: contacting the sample with HCV antigens and anti-HCV coreantibodies attached to a solid phase, adding a polyoxyethylene ether tosaid sample, detecting captured antigens and antibodies by addinglabeled anti-human IgG and labeled anti-HCV core antibodies anddetecting the signal as an indication of the presence of HCV infection.2. The method of claim 1 wherein the polyoxyethylene ether is selectedfrom the group consisting of 2 oleyl ether, 8 stearate, 50 stearate, 3lauryl ether, 4 lauryl ether, 5 lauryl ether, 7 lauryl ether, 8 laurylether, 9 lauryl ether, 10 lauryl ether, 10 cetyl ether, 10 oleyl ether,100 stearate, 20 cetyl ether, 40 stearate, 100 stearyl ether, 20 oleylether, 23 lauryl ether, and combinations thereof.
 3. The method of claim1 wherein the HCV antigens are c200-3, NS-5 and a modified core antigen.4. The method of claim 1 wherein the HCV anitbodies are anti-core murinemonoclonal antibodies.
 5. The method of claim 4 whereing the anti-coremurine monoclonal antibodies are c11-3 and c11-7.
 6. An immunoassay kit,suitable for detecting the presence of HCV in a sample, comprisingamounts sufficient to perform said immunoassay of: (a) HCV captureantigens and antibodies bound to a solid phase, and (b) detectablylabeled anti-human IgG and detectably labeled anti-HCV core antibodies.